The leukocyte specific beta2 integrins are central to the biological function of these cells. Beta2 integrins mediate the divalent metal ion dependent adhesion of leukocytes including homing, firm adhesion, migration, respiratory burst and clearance of pathogens through phagocytosis and cell-mediated killing, beta2 integrins also contribute to injury in many non-infectious diseases, such as renal failure, allograft rejection, heart attacks, strokes and autoimmune diabetic complications, where the receptors are pathologically activated. Therefore, beta2 integrins are important therapeutic targets in inflammation, autoimmunity, and transplantation. Integrins use bi-directional signaling to regulate cellular functions. Inside-out signals activate integrins by inducing conformational changes in their extracellular ligand-binding domains. Structural basis for integrin activation is poorly understood and is a focus of intense current research. Recently described high-resolution alphaVbeta3 integrin crystal structures highlight distinctive features of the integrin dimers and provide new insights into the nature of their activation. Our aim is to develop and test specific, structure-based hypotheses for integrin activation, with a focus on beta2 integrin CD11b/CD18, by using alphaVbeta3 structure as the model for the CD11b/CD18 integrin. We will generate mutations in integrin subunits and study their effect on integrin activation and biological function using cell-based and biochemical assays. In aim 1, we will evaluate the basis of metal ion coordination at alpha-Genu, and determine the role of flexion in allosteric integrin activation. Alpha-genu is the knee structure in the alpha-subunit that lies between the thigh and the calf-1 domains. The structure serves as a highly flexible hinge, and is the site of the bend in the crystal structure that has a divalent metal ion associated with it. In aim 2, we will examine the deadbolt hypothesis and study the role of betaTD in inside-out activation of integrins. Beta tail domain (betaTD) is a novel domain that was found in beta-subunit in the integrin crystal structure. Comparison of unliganded and liganded integrin structures shows that this domain may regulate integrin activation by binding with the betaA domain and thereby acting as a deadbolt to keep integrins in an inactive form. In aim 3, we will determine the function of a extracellular juxta-membrane residue in integrin homo-oligomerization and clustering. Sequence alignment between various integrins shows that there are a number of conserved residues between the betaTD and the transmembrane region. This residue may play a part in integrin activation or clustering. Given the importance of beta2 integrins in every aspect of leukocyte function, these studies will have a profound impact on cell biology and will facilitate design of novel therapeutics to treat the many diseases resulting from pathologic modulation of integrins. The insights gained from these studies are also likely to extend to other integrins.